Salts of the Janus kinase inhibitor (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile

ABSTRACT

The present invention provides salt forms of (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile that are useful in the modulation of Janus kinase activity and are useful in the treatment of diseases related to activity of Janus kinases including, for example, immune-related diseases, skin disorders, myeloid proliferative disorders, cancer, and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/164,518 filed on May 25, 2016, which is a continuation of U.S.application Ser. No. 14/270,915 filed on May 6, 2014, now issued as U.S.Pat. No. 9,376,439, which is a continuation of U.S. application Ser. No.14/097,588, filed on Dec. 5, 2013, now issued as U.S. Pat. No.8,722,693, which is a continuation of U.S. application Ser. No.12/137,892, filed Jun. 12, 2008, which claims the benefit of U.S. Ser.No. 60/943,705, filed Jun. 13, 2007, the disclosure of each of which isincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention provides salt forms of(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilethat are useful in the modulation of Janus kinase activity and areuseful in the treatment of diseases related to activity of Janus kinasesincluding, for example, immune-related diseases, skin disorders, myeloidproliferative disorders, cancer, and other diseases.

BACKGROUND OF THE INVENTION

Protein kinases (PKs) are a group of enzymes that regulate diverse,important biological processes including cell growth, survival anddifferentiation, organ formation and morphogenesis, neovascularization,tissue repair and regeneration, among others. Protein kinases exerttheir physiological functions through catalyzing the phosphorylation ofproteins (or substrates) and thereby modulating the cellular activitiesof the substrates in various biological contexts. In addition to thefunctions in normal tissues/organs, many protein kinases also play morespecialized roles in a host of human diseases including cancer. A subsetof protein kinases (also referred to as oncogenic protein kinases), whendysregulated, can cause tumor formation and growth, and furthercontribute to tumor maintenance and progression (Blume-Jensen P et al,Nature 2001, 411(6835):355-365). Thus far, oncogenic protein kinasesrepresent one of the largest and most attractive groups of proteintargets for cancer intervention and drug development.

The Janus Kinase (JAK) family plays a role in the cytokine-dependentregulation of proliferation and function of cells involved in immuneresponse. Currently, there are four known mammalian JAK family members:JAK1 (also known as Janus kinase-1), JAK2 (also known as Januskinase-2), JAK3 (also known as Janus kinase, leukocyte; JAKL; L-JAK andJanus kinase-3) and TYK2 (also known as protein-tyrosine kinase 2). TheJAK proteins range in size from 120 to 140 kDa and comprise sevenconserved JAK homology (JH) domains; one of these is a functionalcatalytic kinase domain, and another is a pseudokinase domainpotentially serving a regulatory function and/or serving as a dockingsite for STATs (Scott, Godshall et al. 2002, supra).

Blocking signal transduction at the level of the JAK kinases holdspromise for developing treatments for human cancers. Inhibition of theJAK kinases is also envisioned to have therapeutic benefits in patientssuffering from skin immune disorders such as psoriasis, and skinsensitization. Accordingly, inhibitors of Janus kinases or relatedkinases are widely sought and several publications report effectiveclasses of compounds. For example, certain JAK inhibitors, including(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitriledepicted below, are reported in U.S. Ser. No. 11/637,545, filed Dec. 12,2006.

Thus, new or improved forms of existing Janus kinase inhibitors arecontinually needed for developing new, improved, and more effectivepharmaceutical formulations for the treatment of cancer and otherdiseases. The salt forms and methods described herein are directedtoward these needs and other ends.

SUMMARY OF THE INVENTION

The present invention provides, inter alia, salts selected from:

-   (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile    maleic acid salt;-   (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile    sulfuric acid salt; and-   (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile    phosphoric acid salt.

The present invention further provides methods of preparing a salt ofthe invention comprising combining(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilewith maleic acid, sulfuric acid, or phosphoric acid.

The present invention further provides compositions comprising a saltform of the invention and at least one pharmaceutically acceptablecarrier.

The present invention further provides methods of modulating an activityof JAK comprising contacting JAK with a salt of the invention.

The present invention further provides methods of treating a disease ina patient, wherein the disease is associated with JAK activity,comprising administering to the patient a therapeutically effectiveamount of a salt of the invention.

The present invention further provides methods of treating cancer, skindisorders, or inflammation in a patient, comprising administering to thepatient a therapeutically effective amount of a salt of the invention.

DETAILED DESCRIPTION

The present invention provides, inter alia, salts of the JAK inhibitor(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrileselected from the maleic acid salt, sulfuric acid salt, and phosphoricacid salt. These salts modulate the activity of one or more JAKs and areuseful, for example, in the treatment of diseases associated with JAKexpression or activity.

The salts of the invention have numerous advantageous properties overthe free base form and other salt forms. In particular, these salts werehighly crystalline which would facilitate the preparation ofpharmaceutical formulations and improve general handling, manipulation,and storage of the active ingredient. The salts of the invention alsohave superior aqueous solubility, rate of dissolution, chemicalstability (with a longer shelf life), compatibility with excipients, andreproducibility compared with the free base form.

In some embodiments, the salts of the invention are substantiallyisolated. By “substantially isolated” is meant that the salt is at leastpartially or substantially separated from the environment in which itwas formed or detected. Partial separation can include, for example, acomposition enriched in the salt of the invention. Substantialseparation can include compositions containing at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 97%, or at least about 99% byweight of the salt.

Salts of the invention also include all isotopes of atoms occurring inthe salts. Isotopes include those atoms having the same atomic numberbut different mass numbers. For example, isotopes of hydrogen includetritium and deuterium.

Salts of the invention can be prepared using known techniques.Conventionally, a salt form is prepared by combining in solution thefree base compound and an acid containing the anion of the salt formdesired, and then isolating the solid salt product from the reactionsolution (e.g., by crystallization, precipitation, evaporation, etc.).Other salt-forming techniques can be employed.

Methods of Use

Salts of the invention can modulate activity of one or more Januskinases (JAKs). The term “modulate” is meant to refer to an ability toincrease or decrease the activity of one or more members of the JAKfamily of kinases. Accordingly, compounds of the invention can be usedin methods of modulating a JAK by contacting the JAK with any one ormore of the compounds or compositions described herein. In someembodiments, salts of the present invention can act as inhibitors of oneor more JAKs. In some embodiments, compounds of the present inventioncan act to stimulate the activity of one or more JAKs. In furtherembodiments, the compounds of the invention can be used to modulateactivity of a JAK in an individual in need of modulation of the receptorby administering a modulating amount of a salt of the invention.

JAKs to which the present salts bind and/or modulate include any memberof the JAK family. In some embodiments, the JAK is JAK1, JAK2, JAK3 orTYK2. In some embodiments, the JAK is JAK1 or JAK2. In some embodiments,the JAK is JAK2. In some embodiments, the JAK is JAK3.

The salts of the invention can be selective. By “selective” is meantthat the compound binds to or inhibits a JAK with greater affinity orpotency, respectively, compared to at least one other JAK. In someembodiments, the compounds of the invention are selective inhibitors ofJAK1 or JAK2 over JAK3 and/or TYK2. In some embodiments, the salts ofthe invention are selective inhibitors of JAK2 (e.g., over JAK1, JAK3and TYK2). Without wishing to be bound by theory, because inhibitors ofJAK3 can lead to immunosuppressive effects, a compound which isselective for JAK2 over JAK3 and which is useful in the treatment ofcancer (such as multiple myeloma, for example) can offer the additionaladvantage of having fewer immunosuppressive side effects. Selectivitycan be at least about 5-fold, 10-fold, at least about 20-fold, at leastabout 50-fold, at least about 100-fold, at least about 200-fold, atleast about 500-fold or at least about 1000-fold. Selectivity can bemeasured by methods routine in the art. In some embodiments, selectivitycan be tested at the Km of each enzyme. In some embodiments, selectivityof salts of the invention for JAK2 over JAK3 can be determined by thecellular ATP concentration.

Another aspect of the present invention pertains to methods of treatinga JAK-associated disease or disorder in an individual (e.g., patient) byadministering to the individual in need of such treatment atherapeutically effective amount or dose of a salt of the presentinvention or a pharmaceutical composition thereof. A JAK-associateddisease can include any disease, disorder or condition that is directlyor indirectly linked to expression or activity of the JAK, includingoverexpression and/or abnormal activity levels. A JAK-associated diseasecan also include any disease, disorder or condition that can beprevented, ameliorated, or cured by modulating JAK activity.

Examples of JAK-associated diseases include diseases involving theimmune system including, for example, organ transplant rejection (e.g.,allograft rejection and graft versus host disease).

Further examples of JAK-associated diseases include autoimmune diseasessuch as multiple sclerosis, rheumatoid arthritis, juvenile arthritis,type I diabetes, lupus, psoriasis, inflammatory bowel disease,ulcerative colitis, Crohn's disease, myasthenia gravis, immunoglobulinnephropathies, autoimmune thyroid disorders, and the like. In someembodiments, the autoimmune disease is an autoimmune bullous skindisorder such as pemphigus vulgaris (PV) or bullous pemphigoid (BP).

Further examples of JAK-associated diseases include allergic conditionssuch as asthma, food allergies, atopic dermatitis and rhinitis. Furtherexamples of JAK-associated diseases include viral diseases such asEpstein Barr Virus (EBV), Hepatitis B, Hepatitis C, HIV, HTLV 1,Varicella-Zoster Virus (VZV) and Human Papilloma Virus (HPV).

Further examples of JAK-associated diseases or conditions include skindisorders such as psoriasis (for example, psoriasis vulgaris), atopicdermatitis, skin rash, skin irritation, skin sensitization (e.g.,contact dermatitis or allergic contact dermatitis). For example, certainsubstances including some pharmaceuticals when topically applied cancause skin sensitization. In some embodiments, co-administration orsequential administration of at least one JAK inhibitor of the inventiontogether with the agent causing unwanted sensitization can be helpful intreating such unwanted sensitization or dermatitis. In some embodiments,the skin disorder is treated by topical administration of at least oneJAK inhibitor of the invention.

In further embodiments, the JAK-associated disease is cancer includingthose characterized by solid tumors (e.g., prostate cancer, renalcancer, hepatic cancer, pancreatic cancer, gastric cancer, breastcancer, lung cancer, cancers of the head and neck, thyroid cancer,glioblastoma, Kaposi's sarcoma, Castleman's disease, melanoma etc.),hematological cancers (e.g., lymphoma, leukemia such as acutelymphoblastic leukemia, acute myelogenous leukemia (AML), or multiplemyeloma), and skin cancer such as cutaneous T-cell lymphoma (CTCL) andcutaneous B-cell lymphoma. Example cutaneous T-cell lymphomas includeSezary syndrome and mycosis fungoides.

JAK-associated diseases can further include those characterized byexpression of a mutant JAK2 such as those having at least one mutationin the pseudo-kinase domain (e.g., JAK2V617F).

JAK-associated diseases can further include myeloproliferative disorders(MPDs) such as polycythemia vera (PV), essential thrombocythemia (ET),myeloid metaplasia with myelofibrosis (MMM), chronic myelogenousleukemia (CML), chronic myelomonocytic leukemia (CMML),hypereosinophilic syndrome (HES), systemic mast cell disease (SMCD), andthe like.

Further JAK-associated diseases include inflammation and inflammatorydiseases. Example inflammatory diseases include inflammatory diseases ofthe eye (e.g., iritis, uveitis, scleritis, conjunctivitis, or relateddisease), inflammatory diseases of the respiratory tract (e.g., theupper respiratory tract including the nose and sinuses such as rhinitisor sinusitis or the lower respiratory tract including bronchitis,chronic obstructive pulmonary disease, and the like), inflammatorymyopathy such as myocarditis, and other inflammatory diseases. Otherinflammatory diseases treatable by the compounds of the inventioninclude systemic inflammatory response syndrome (SIRS) and septic shock.

The JAK inhibitors described herein can further be used to treatischemia reperfusion injuries or a disease or condition related to aninflammatory ischemic event such as stroke or cardiac arrest. The JAKinhibitors described herein can further be used to treat anorexia,cachexia, or fatigue such as that resulting from or associated withcancer. The JAK inhibitors described herein can further be used to treatrestenosis, sclerodermitis, or fibrosis. The JAK inhibitors describedherein can further be used to treat conditions associated with hypoxiaor astrogliosis such as, for example, diabetic retinopathy, cancer, orneurodegeneration. See, e.g., Dudley, A. C. et al. Biochem. J. 2005, 390(Pt 2):427-36 and Sriram, K. et al. J. Biol. Chem. 2004,279(19):19936-47. Epub 2004 Mar. 2.

The JAK inhibitors described herein can further be used to treat goutand increased prostate size due to, e.g., benign prostatic hypertrophyor benign prostatic hyperplasia.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a JAK with a salt of the invention includes theadministration of a salt of the present invention to an individual orpatient, such as a human, having a JAK, as well as, for example,introducing a salt of the invention into a sample containing a cellularor purified preparation containing the JAK.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active salt or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) preventing the disease; for example, preventing a disease,condition or disorder in an individual who may be predisposed to thedisease, condition or disorder but does not yet experience or displaythe pathology or symptomatology of the disease; (2) inhibiting thedisease; for example, inhibiting a disease, condition or disorder in anindividual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder; and (3)ameliorating the disease; for example, ameliorating a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,reversing the pathology and/or symptomatology) such as decreasing theseverity of disease.

Combination Therapies

One or more additional pharmaceutical agents such as, for example,chemotherapeutics, anti-inflammatory agents, steroids,immunosuppressants, as well as Bcr-Abl, Flt-3, RAF and FAK kinaseinhibitors such as, for example, those described in WO 2006/056399, orother agents can be used in combination with the salts of the presentinvention for treatment of JAK-associated diseases, disorders orconditions. The one or more additional pharmaceutical agents can beadministered to a patient simultaneously or sequentially.

Example chemotherapeutics include proteosome inhibitors (e.g.,bortezomib), thalidomide, revlimid, and DNA-damaging agents such asmelphalan, doxorubicin, cyclophosphamide, vincristine, etoposide,carmustine, and the like.

Example steroids include coriticosteroids such as dexamethasone orprednisone.

Example Bcr-Abl inhibitors include the compounds, and pharmaceuticallyacceptable salts thereof, of the genera and species disclosed in U.S.Pat. No. 5,521,184, WO 04/005281, EP2005/009967, EP2005/010408, and U.S.Ser. No. 60/578,491.

Example suitable Flt-3 inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 03/037347, WO03/099771, and WO 04/046120.

Example suitable RAF inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO05/028444.

Example suitable FAK inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 04/080980, WO04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402.

In some embodiments, the salt forms of the invention can be used incombination with other kinase inhibitors such as imatinib, particularlyfor the treatment of patients resistant to imatinib or other kinases.

In some embodiments, one or more salt forms of the invention can be usedin combination with a chemotherapeutic in the treatment of cancer, suchas multiple myeloma, and may improve the treatment response as comparedto the response to the chemotherapeutic agent alone, withoutexacerbation of its toxic effects. Examples of additional pharmaceuticalagents used in the treatment of multiple myeloma, for example, caninclude, without limitation, melphalan, melphalan plus prednisone [MP],doxorubicin, dexamethasone, and Velcade (bortezomib). Further additionalagents used in the treatment of multiple myeloma include Bcr-Abl, Flt-3,RAF and FAK kinase inhibitors. Additive or synergistic effects aredesirable outcomes of combining a JAK inhibitor of the present inventionwith an additional agent. Furthermore, resistance of multiple myelomacells to agents such as dexamethasone may be reversible upon treatmentwith a JAK inhibitor of the present invention. The agents can becombined with the present compounds in a single or continuous dosageform, or the agents can be administered simultaneously or sequentiallyas separate dosage forms.

In some embodiments, a corticosteroid such as dexamethasone isadministered to a patient in combination with at least one JAK inhibitorwhere the dexamethasone is administered intermittently as opposed tocontinuously.

In some further embodiments, combinations of one or more JAK inhibitorsof the invention with other therapeutic agents can be administered to apatient prior to, during, and/or after a bone marrow transplant or stemcell transplant.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the salts of the invention can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical (includingtransdermal, epidermal, ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer;intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal, intramuscular or injection or infusion; orintracranial, e.g., intrathecal or intraventricular, administration.Parenteral administration can be in the form of a single bolus dose, ormay be, for example, by a continuous perfusion pump. Pharmaceuticalcompositions and formulations for topical administration may includetransdermal patches, ointments, lotions, creams, gels, drops,suppositories, sprays, liquids and powders. Conventional pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable. Coated condoms, gloves and the like may also beuseful.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds of the inventionabove in combination with one or more pharmaceutically acceptablecarriers (excipients). In making the compositions of the invention, theactive ingredient is typically mixed with an excipient, diluted by anexcipient or enclosed within such a carrier in the form of, for example,a capsule, sachet, paper, or other container. When the excipient servesas a diluent, it can be a solid, semi-solid, or liquid material, whichacts as a vehicle, carrier or medium for the active ingredient. Thus,the compositions can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,syrups, aerosols (as a solid or in a liquid medium), ointmentscontaining, for example, up to 10% by weight of the active compound,soft and hard gelatin capsules, suppositories, sterile injectablesolutions, and sterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention can beprepared by processes known in the art, for example see InternationalPatent Application No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face masks tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theformulation in an appropriate manner.

The amount of salt or composition administered to a patient will varydepending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the salts of the present invention can varyaccording to, for example, the particular use for which the treatment ismade, the manner of administration of the compound, the health andcondition of the patient, and the judgment of the prescribing physician.The proportion or concentration of a salt of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the salts of the invention canbe provided in an aqueous physiological buffer solution containing about0.1 to about 10% w/v of the compound for parenteral administration. Sometypical dose ranges are from about 1 μg/kg to about 1 g/kg of bodyweight per day. In some embodiments, the dose range is from about 0.01mg/kg to about 100 mg/kg of body weight per day. The dosage is likely todepend on such variables as the type and extent of progression of thedisease or disorder, the overall health status of the particularpatient, the relative biological efficacy of the compound selected,formulation of the excipient, and its route of administration. Effectivedoses can be extrapolated from dose-response curves derived from invitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents such as a chemotherapeutic, steroid,anti-inflammatory compound, or immunosuppressant, examples of which arelisted hereinabove.

Labeled Compounds and Assay Methods

Another aspect of the present invention relates to labeled salts of theinvention (radio-labeled, fluorescent-labeled, etc.) that would beuseful not only in imaging techniques but also in assays, both in vitroand in vivo, for localizing and quantitating JAK in tissue samples,including human, and for identifying JAK ligands by inhibition bindingof a labeled compound. Accordingly, the present invention includes JAKassays that contain such labeled compounds.

The present invention further includes isotopically-labeled salts of theinvention. An “isotopically” or “radio-labeled” compound is a salt ofthe invention where one or more atoms are replaced or substituted by anatom having an atomic mass or mass number different from the atomic massor mass number typically found in nature (i.e., naturally occurring).Suitable radionuclides that may be incorporated in compounds of thepresent invention include but are not limited to ²H (also written as Dfor deuterium), ³H (also written as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N,¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I,¹²⁵I and ¹³¹I. The radionuclide that is incorporated in the instantradio-labeled compounds will depend on the specific application of thatradio-labeled compound. For example, for in vitro metalloproteaselabeling and competition assays, compounds that incorporate ³H, ¹⁴C,⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S or will generally be most useful. Forradio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Bror ⁷⁷Br will generally be most useful.

It is understood that a “radio-labeled” or “labeled compound” is a saltthat has incorporated at least one radionuclide. In some embodiments theradionuclide is selected from the group consisting of ³H, ¹⁴C, ¹²⁵I, ³⁵Sand ⁸²Br.

The present invention can further include synthetic methods forincorporating radio-isotopes into compounds of the invention. Syntheticmethods for incorporating radio-isotopes into organic compounds are wellknown in the art, and a person of ordinary skill in the art will readilyrecognize the methods applicable for the compounds of invention.

A labeled salt of the invention can be used in a screening assay toidentify/evaluate compounds. For example, a newly synthesized oridentified compound (i.e., test compound) which is labeled can beevaluated for its ability to bind a JAK by monitoring its concentrationvariation when contacting with the JAK, through tracking of thelabeling. For example, a test compound (labeled) can be evaluated forits ability to reduce binding of another compound which is known to bindto a JAK (i.e., standard compound). Accordingly, the ability of a testcompound to compete with the standard compound for binding to the JAKdirectly correlates to its binding affinity. Conversely, in some otherscreening assays, the standard compound is labeled and test compoundsare unlabeled. Accordingly, the concentration of the labeled standardcompound is monitored in order to evaluate the competition between thestandard compound and the test compound, and the relative bindingaffinity of the test compound is thus ascertained.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of JAK-associated diseases ordisorders, such as cancer, inflammation, or skin disorders, whichinclude one or more containers containing a pharmaceutical compositioncomprising a therapeutically effective amount of a salt of theinvention. Such kits can further include, if desired, one or more ofvarious conventional pharmaceutical kit components, such as, forexample, containers with one or more pharmaceutically acceptablecarriers, additional containers, etc., as will be readily apparent tothose skilled in the art. Instructions, either as inserts or as labels,indicating quantities of the components to be administered, guidelinesfor administration, and/or guidelines for mixing the components, canalso be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

EXAMPLES Example 1: Preparation of(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilemaleic acid salt

To a test tube was added(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile(153.7 mg, 0.5 mmol) and maleic acid (61.7 mg) followed by isopropylalcohol (IPA) (4 mL). The resulting mixture was heated to clear, cooledto room temperature, and then stirred for another 2.5 hours. Theprecipitate was collected by filtration and the cake was washed with 0.8mL of cold IPA. The cake was dried under vacuum to constant weight toprovide the final salt product (173 mg).

The maleic acid salt was shown to be a 1:1 salt by H¹ NMR andcrystallinity was confirmed by X-ray powder diffraction (XRPD).Differential scanning calorimetry (DSC) gave a sharp melting peak atabout 175.96° C. (onset at 175.67° C.). The product showed only slightweight loss up to 150° C. by thermogravimetric analysis (TGA).

Example 2: Preparation of(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilephosphoric acid salt

To a test tube was added(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile(153.5 mg) and phosphoric acid (56.6 mg) followed by isopropyl alcohol(IPA) (5.75 mL). The resulting mixture was heated to clear, cooled toroom temperature, and then stirred for another 2 hours. The precipitatewas collected by filtration and the cake was washed with 0.6 mL of coldIPA. The cake was dried under vacuum to constant weight to provide thefinal salt product (171.7 mg).

The phosphroic acid salt was shown to be a 1:1 salt by ¹H NMR andcrystallinity was confirmed by X-ray powder diffraction (XRPD).Differential scanning calorimetry (DSC) gave a sharp melting peak atabout 198.66° C. The product showed little weight loss up to 200° C. byTGA.

Example 3: Preparation of(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilesulfuric acid salt

To a test tube was added(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile(153.0 mg) and sulfuric acid (56.1 mg) followed by acetonitrile (7.0mL). The resulting mixture was heated to clear, cooled to roomtemperature, and then stirred for another 2 hours. The precipitate wascollected by filtration and the cake was washed with 0.8 mL of coldacetonitrile. The cake was dried under vacuum to constant weight toprovide the final salt product (180 mg).

The sulfuric acid salt was shown to be a 1:1 salt by ¹H NMR andcrystallinity was confirmed by X-ray powder diffraction (XRPD).Differential scanning calorimetry (DSC) gave a sharp melting peak atabout 186.78° C. The product showed little weight loss up to 175° C. byTGA.

Example A

In Vitro JAK Kinase Assay

Inhibitory activity of test compounds on JAK targets can be testedaccording to the following in vitro assay described in Park et al.,Analytical Biochemistry 1999, 269, 94-104. The catalytic domains ofhuman JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a.781-1124) with an N-terminal His tag are expressed using baculovirus ininsect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3is assayed by measuring the phosphorylation of a biotinylated peptide.The phosphorylated peptide was detected by homogenous time resolvedfluorescence (HTRF). IC₅₀s of compounds are measured for each kinase inthe reactions that contain the enzyme, ATP and 500 nM peptide in 50 mMTris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%)BSA. The ATP concentration in the reactions is 90 μM for Jak1, 30 μM forJak2 and 3 μM for Jak3. Reactions are carried out at room temperaturefor 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nMEu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to theEuropium labeled antibody takes place for 40 minutes and HTRF signal ismeasured on a Fusion plate reader (Perkin Elmer, Boston, Mass.). Boththe phosphoric acid salt of the invention, and the corresponding freebase compound, were found to have IC₅₀ values of less than 50 nM foreach of JAK1, JAK2, and JAK3.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method of inhibiting JAK1 and/or JAK2 in apatient suffering from an autoimmune skin disease, comprisingadministering to said patient a therapeutically effective amount of acompound that is(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilephosphoric acid salt.
 2. The method of claim 1, wherein said autoimmuneskin disease is bullous skin disorder.
 3. The method of claim 2, whereinsaid bullous skin disorder is pemphigus vulgaris (PV) or bullouspemphigoid (BP).
 4. The method of claim 3, wherein said bullous skindisorder is pemphigus vulgaris (PV).
 5. The method of claim 1, whereinthe autoimmune skin disease is atopic dermatitis, psoriasis, skinsensitization, skin irritation, skin rash, contact dermatitis, orallergic contact sensitization.
 6. The method of claim 5, wherein theautoimmune skin disease is psoriasis.
 7. The method of claim 5, whereinthe autoimmune skin disease is atopic dermatitis.
 8. The method of claim1, wherein(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilephosphoric acid salt is a crystalline salt.
 9. The method of claim 8,wherein said crystalline salt is a 1:1(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile:phosphoricacid salt.
 10. A method of treating an autoimmune skin disease selectedfrom pemphigus vulgaris (PV), bullous pemphigoid (BP), atopicdermatitis, and psoriasis in a patient, comprising administering to saidpatient a therapeutically effective amount of a compound that is(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilephosphoric acid salt, wherein said treating refers to ameliorating orinhibiting the disease in a patient.
 11. The method of claim 10, whereinthe autoimmune skin disorder is pemphigus vulgaris (PV) or bullouspemphigoid (BP).
 12. The method of claim 10, wherein the autoimmune skindisorder is pemphigus vulgaris (PV).
 13. The method of claim 10, whereinthe autoimmune skin disease is psoriasis.
 14. The method of claim 10,wherein the autoimmune skin disease is atopic dermatitis.
 15. The methodof claim 10, wherein(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilephosphoric acid salt is a crystalline salt.
 16. The method of claim 15,wherein said crystalline salt is a 1:1(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile:phosphoricacid salt.
 17. A method of treating an autoimmune skin disease selectedfrom pemphigus vulgaris (PV), bullous pemphigoid (BP), atopicdermatitis, and psoriasis in a patient, comprising administering to saidpatient a therapeutically effective amount of a pharmaceuticalcomposition comprising(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilephosphoric acid salt and a pharmaceutically acceptable carrier, whereinsaid treating refers to ameliorating or inhibiting the disease in apatient.
 18. The method of claim 17, wherein the autoimmune skindisorder is pemphigus vulgaris (PV).
 19. The method of claim 17, whereinthe autoimmune skin disease is psoriasis.
 20. The method of claim 17,wherein the autoimmune skin disease is atopic dermatitis.
 21. The methodof claim 17, wherein the pharmaceutical composition is suitable for oraladministration.
 22. The method of claim 17, wherein the pharmaceuticalcomposition is suitable for topical administration.
 23. The method ofclaim 17, wherein(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilephosphoric acid salt is a crystalline salt.
 24. The method of claim 23,wherein said crystalline salt is a 1:1(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile:phosphoricacid salt.
 25. A method of treating atopic dermatitis in a patient,comprising topically administering to said patient a therapeuticallyeffective amount of a pharmaceutical composition comprising(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilephosphoric acid salt and a pharmaceutically acceptable carrier, whereinsaid treating refers to ameliorating or inhibiting the disease in apatient.
 26. The method of claim 1, wherein said salt is administeredtopically.
 27. The method of claim 10, wherein said salt is administeredtopically.
 28. The method of claim 17, wherein said pharmaceuticalcomposition is administered topically.